HIER uses a heating agent like a microwave, steamer, or pressure cooker and is a milder process for epitope exposure than PIER. For HIER, tissues are placed in a specific buffer, often citrate or Tris-EDTA, and then heated to 95–100°C using one of the mechanisms listed previously for about 20 minutes.
Citrate antigen retrieval buffer is made from sodium citrate or citric acid and is typically used at pH 6. Sometimes a detergent like Tween-20 is added to citrate buffer to increase efficacy of the buffer. Citrate buffer is commonly used because it does not disrupt tissue morphology.
Tris-EDTA antigen retrieval buffer is made of Tris base and EDTA and is typically used at pH 9. This alkaline buffer is used for antigens that are harder to detect, and it can be more effective at unmasking phosphoproteins than citrate buffer. However, since Tris-EDTA is more damaging to tissue, there can be distorted tissue morphology or loss of regions from the slide. Additionally, Tris-EDTA can increase background and off-target staining.
While it is well known that antigen retrieval can greatly enhance the outcomes of immunohistochemistry, there is not a universal process or buffer used. This means that researchers need to determine optimal retrieval conditions including buffer, pH, heat, and proteolytic conditions experimentally. Many antibody suppliers will provide retrieval recommendations for each antibody, so make sure to use this information as a starting point for your optimization.
